Introduction

Perturbations in lipid metabolism are associated with insulin resistance and type 2 diabetes (T2D) (1). An increased lipid supply, altered lipid partitioning, and reduced capacity for skeletal muscle fat oxidation have been
proposed to contribute to intramyocellular lipid (IMCL) accumulation, lipotoxicity, and insulin resistance (2–4). However, whether these characteristics of T2D are inherited or simply the consequence of an altered lifestyle and obesity
remains an ongoing debate. In that context, studies in human primary myotubes are of interest because cell autonomous models
are devoid of direct environmental influences. Hulver et al. (5) used such a model to demonstrate that myotubes of nondiabetic, severely obese individuals retain abnormal lipid partitioning
(i.e., elevated triacylglycerol [TAG]-to-blunted fatty acid [FA] oxidation ratio) (5). Of note, Aguer et al. (6) showed that T2D subjects had increased IMCL content in muscle tissue and that this elevation was maintained in primary myotubes
from these subjects compared with obese nondiabetic control subjects, indicating that increased IMCL content is preserved
in vitro. However, the underlying reason for this finding was not examined. Elevated FA uptake could contribute to an increased
IMCL content observed in obesity and T2D. Some studies showed no difference in uptake of FAs (7,8), whereas others showed increased FA uptake in skeletal muscle of obese and T2D individuals (9). Likewise, studies in primary human myotubes showed inconsistent results (6,10,11). Taken together, whether disturbances that exist in skeletal muscle tissue lipid metabolism of T2D individuals are preserved
in the myotubes is unclear. If these disturbances persist in the myotubes, are they related to any aspects of the in vivo
metabolic phenotype of the donors?

Because aberrant lipid metabolism is a central feature of obesity and T2D, the goal of the current study was to investigate
whether disturbances exist in intramyocellular FA metabolism in skeletal muscle of obese T2D individuals compared with BMI-
and age-matched normoglycemic control subjects and to examine whether disturbances in lipid metabolism are retained in vitro
in the myotubes established from these donors. This study is a unique combination of ex vivo and in vitro analyses of lipid
metabolism in human skeletal muscle coupled with detailed in vivo clinical phenotyping to assess insulin sensitivity and aerobic
capacity. We measured ex vivo FA metabolism in skeletal muscle tissue and established primary myotubes from these donors for
in vitro studies. For comparison, we measured FA metabolism in skeletal muscle tissue of young, lean males. The myotubes were
used to investigate lipid turnover (FA uptake, oxidation, and storage) using two different long-chain FAs. We show that FA
incorporation into TAG is impaired in T2D muscle and in the myotubes established from T2D donors, indicating that this perturbation
is inherent in the T2D muscle cell.

Research Design and Methods

Participants

Twelve obese males with T2D (n = 6) and BMI- and age-matched males (controls) participated. Participants with T2D were given a diagnosis at least 1 year
before the study, were noninsulin dependent, had well-controlled diabetes (HbA1c < 7.8%; 62 mmol/mol; 177 mg/dL), and had no diabetes-related comorbidities. Medication use (metformin only or metformin plus
sulfonylureas) was stable for at least 6 months. Controls had no family history of diabetes. We included data from 16 young,
lean males who participated in another (unpublished) study. The studies were approved by the Medical Ethical Committee of
Maastricht University. All participants gave written informed consent, performed a maximal aerobic capacity test (VO2max) (12), and underwent dual-energy X-ray absorptiometry or hydrostatic weighing for body composition (13).

Hyperinsulinemic-Euglycemic Clamp

To measure peripheral insulin sensitivity, a two-step hyperinsulinemic-euglycemic clamp was performed according to DeFronzo
et al. (14). Briefly, after an overnight fast, a blood sample was drawn to measure glucose, insulin, and free FA levels. Step 1 was
initiated with an insulin infusion at 10 mU/m2/min for 4 h with variable coinfusion of 20% glucose. Step 2 consisted of a 2-h insulin infusion at 40 mU/m2/min (20% variable glucose). The M-value was calculated as the glucose infusion rate and corrected for fat-free mass (FFM).

Muscle Biopsy

Muscle biopsy specimens were taken from the vastus lateralis according to Bergstrom (15) and processed the same day for ex vivo assays and cell culture. Remaining tissue was stored at −80°C for future analyses.

Primary Muscle Cell Cultures

Primary skeletal muscle cell cultures were established as previously described (16). Briefly, satellite cells were isolated and grown in media supplemented with 16% FBS at 37°C and 5% CO2.

Real-Time Quantitative RT-PCR

Total RNA was isolated from ∼20 mg of muscle tissue as previously described (17). Primers and probes are shown in Supplementary Table 1. Real-time quantitative RT-PCR was performed as one-step reactions (18) as previously described (19). All expression data were normalized by dividing the target gene by the internal control gene.

Oil Red O Staining

Fresh muscle cryosections were stained for IMCL by Oil Red O as described previously (20) and expressed per cell surface area.

14C-Labeled Ex Vivo Palmitate Oxidation and Lipid Incorporation

Palmitate oxidation was determined by measuring production of 14CO2 and acid-soluble metabolites (14C-ASMs) in skeletal muscle homogenates containing 250 mmol/L sucrose, 10 mmol/L Tris-HCl, 1 mmol/L EDTA, and 2 mmol/L ATP.
Reactions were initiated with 0.2 mmol/L palmitate and 0.0175 mmol/L [1-14C]-palmitate and terminated with 70% perchloric acid. CO2 was trapped in 1N NaOH (21,22). All in vitro experiments described next were performed in triplicate per participant and normalized to protein content.

In Vitro 14C-Palmitate Oxidation and Lipid Incorporation

Palmitate oxidation was measured as production of 14CO2 and 14C-ASMs from [1-14C]-palmitate (1 μCi/mL), nonlabeled palmitate (100 μmol/L), and 1 mmol/L carnitine after 3 h. Lipids were extracted from the
myotubes, and lipid incorporation was measured by thin-layer chromatography. Bands corresponding to TAG and diacylglycerol
(DAG) were quantified by liquid scintillation as previously described (16).

In Vitro TAG Levels

Intracellular TAG levels in differentiated myotubes were measured using the method of Schwartz and Wolins (24).

Acyl-CoA:DAG Acyltransferase Activity Assay

Determination of acyl-CoA:DAG acyltransferase (DGAT) activity was performed in cellular homogenates of the human myotubes
as previously described (25). DGAT1 inhibitor was provided by Dr. Robert V. Farese Jr. (26). Data are presented as the rate of formation of 14C-TAG.

Statistics

Results are presented as mean ± SEM. Statistical analyses were performed using SPSS version 16.0 for MacOS 16.0 (IBM Corp.,
Chicago, IL). Statistical comparisons between conditions were performed using unpaired t tests. In the case of in vitro lipid incorporation and corresponding protein expressions, a one-sided unpaired t test was performed because a reduced incorporation in T2D myotubes was not plausible based on the ex vivo data of lipid incorporation.
Pearson correlation coefficients were used to describe the linear association between variables. P < 0.05 was considered statistically significant.

We next measured the incorporation of 14C-palmitate into lipids in skeletal muscle homogenates. Although incorporation of 14C-palmitate into the total lipid pool was similar between the two groups (Fig. 2D), we observed decreased incorporation of 14C-palmitate into TAGs in individuals with T2D (controls: 0.045 ± 0.013 nmol/mg protein; T2D: 0.011 ± 0.002 nmol/mg protein;
P = 0.047) (Fig. 2E), with similar incorporation into DAGs (Fig. 2F). This finding may indicate a blunted ability of the skeletal muscle of individuals with T2D to efficiently store and retain
the lipids in the TAG pool.

In Vitro

Exogenous FA Metabolism

In contrast to 14C-palmitate oxidation in muscle homogenates, in vitro 14C-palmitate oxidation to CO2 was not significantly lower in individuals with T2D (P = 0.38) (Fig. 3A). Furthermore, oxidation to ASMs was not significantly different (P = 0.59) (Fig. 3B) and, consequently, neither was CO2:ASMs (P = 0.38) (Fig. 3C). Moreover, in vitro 14C-palmitate oxidation to CO2 did not correlate with the ex vivo 14C-palmitate oxidation (P = 0.721) (data not shown), suggesting that the capacity to oxidize palmitate to CO2 is not an intrinsic property of the myotubes established from these obese T2D and nondiabetic individuals.

Following a 24-h incubation with 14C-oleate, oxidation was initiated by the addition of carnitine and measured after 3 h. Despite a reduced 14C-oleate incorporation into TAG in myotubes from individuals with T2D, the oxidation of endogenous 14C-oleate to CO2 was not significantly different between the groups (P = 0.384) (Fig. 4D). Likewise, oxidation to ASMs was similar between the two groups (controls: 0.070 ± 0.012 nmol/mg protein; T2D: 0.058 ± 0.015
nmol/mg protein; P = 0.543) (Fig. 4E). Consequently, no differences were observed in CO2:ASMs (P = 0.49) (Fig. 4F). Thus, in concert with the oxidation rates of exogenous 14C-palmitate, oxidation of the endogenously labeled 14C-oleate pool was similar in primary myotubes from both groups.

Of interest, in vivo basal glucose concentration was inversely related to in vitro incorporation of the endogenously labeled
lipid (14C-oleate) into the total lipid pool (r = −0.615, P = 0.033) (Fig. 5A) as well as TAG (r = −0.580, P = 0.048) (Fig. 5B). Insulin sensitivity was also positively associated with 14C-oleate incorporation in the total lipid pool (r = 0.602, P = 0.038) (Fig. 5C). It is important to note that there was no difference in the basal myocellular lipid content between the two donor groups
and, thus, no label dilution effect on the metabolic assays was observed (data not shown).

Correlations between in vivo measures of glucose and insulin sensitivity and in vitro endogenous lipid incorporation. Primary
myotubes were loaded with 400 μmol/L oleate for 24 h and then pulsed in the presence of 1 mmol/L carnitine for 3 h. Incorporation
of 14C-oleate was measured after 24-h incubation with 400 μmol/L oleate in the absence of carnitine. Basal plasma glucose levels
were inversely related to total lipid synthesis (A) and TAG synthesis (B). Insulin sensitivity (M-value at a 40 mU/m2/min insulin infusion rate) was positively associated with total lipid synthesis (C).

Finally, we measured the expressions of the lipid droplet coat proteins PLIN5 and PLIN3 as well as the lipolytic protein ATGL
before and after 24 h of a 400 µmol/L oleate load (Fig. 6E–G). We were unable to detect PLIN2 protein in the myotubes. In contrast to the ex vivo findings, PLIN5 was not different between
the groups in the basal condition. However, PLIN5 increased with the FA load in the control group only (controls: P = 0.073; T2D: P = 0.468) (Fig. 6E). PLIN3 and ATGL protein expressions were not different between groups in the basal condition or significantly changed in
response to the oleate load (Fig. 6F–G). Of note, PLIN5 protein (postoleate load) was significantly associated with the M-value at 40 mU of insulin (r = 0.61, P = 0.035) (data not shown). We further examined whether differences between groups were present in the mRNA expression of
genes involved in lipid metabolism (Supplementary Table 2) in skeletal muscle tissue and/or primary myotubes. Although no significant differences were observed, PGC1α mRNA was significantly
associated with increased palmitate and oleate incorporation into TAG in the myotubes as well as with DGAT activity, whereas
PGC1α mRNA in the muscle tissue was significantly associated with tissue PLIN5 mRNA (Supplementary Table 3).

Ex vivo and in vitro protein expressions. Lipid droplet coating proteins PLIN5 (A), PLIN3 (B), and PLIN2 (C) as well as the lipolytic protein ATGL (D) were measured in the muscle tissues of the control and T2D individuals. Primary myotubes were loaded with 400 μmol/L oleate
for 24 h and then harvested for protein. E–G: Protein expressions were measured before (Basal) and after the 24-h incubation with 400 μmol/L oleate in the absence of
carnitine (OA). E: PLIN5. F: PLIN3. G: ATGL. Sr-actin was used as an internal control for all Western blots. Error bars represent SEM. C, controls.

Discussion

Obesity and T2D are associated with ectopic lipid accumulation in tissues such as skeletal muscle (1,30–33). IMCL content inversely correlates with peripheral insulin sensitivity (34–37), suggesting that fat accumulation leads to insulin resistance. However, studies have shown that lipid intermediates—not
total IMCL per se—are the true culprits of the development of insulin resistance (38,39). Other studies have even suggested that increasing the TAG storage capacity, specifically in skeletal muscle, may be beneficial
(40–42). Although these studies used different methods (i.e., genetic manipulations, exercise), they clearly demonstrated a beneficial
effect of increased TAG storage on skeletal muscle insulin sensitivity. In the current study, we investigated the oxidative
and storage capacities of both skeletal muscle tissue and myotubes established from satellite cells of obese individuals with
T2D and BMI-matched nondiabetic controls. We show that oxidation and incorporation of exogenously supplied long-chain FAs
(palmitate) into TAG is significantly blunted in muscle tissue from individuals with T2D. Of importance, we demonstrate that
this blunted FA oxidative capacity is not retained in primary myotubes from these patients. However, myotubes of individuals
with T2D show reduced incorporation of exogenous palmitate into TAG, and this is significantly related to the impaired palmitate
incorporation into TAG observed ex vivo. Moreover, incorporation of oleate into the total neutral lipid pool, specifically
the TAG pool, after prolonged FA incubation in the absence of carnitine supplementation is significantly reduced in the myotubes
from individuals with T2D. Because the absence of carnitine prevents FA oxidation, this measurement mainly reflects unidirectional
TAG synthesis. Therefore, we conclude that lower lipid incorporation is an intrinsic metabolic characteristic of skeletal
muscle of obese individuals with T2D compared with BMI-matched controls.

We show a blunted complete oxidation of palmitate in the skeletal muscle tissue of obese individuals with T2D compared with
BMI-matched controls and young, lean subjects. Because there were no differences in the ASMs, the ratio of complete to incomplete
oxidation was also reduced in individuals with T2D. These findings extend those of Hulver et al. (43), who demonstrated aberrant exogenous FA oxidation in the skeletal muscle of obese and severely obese (BMI 53.8 ± 3.5 kg/m2) individuals compared with lean control subjects. In this context, the current data imply that T2D is associated with reduced
tissue FA oxidation independent of BMI. However, we did not observe significant differences in oxidation of exogenous palmitate
to CO2 and ASMs between myotubes of obese individuals with T2D and BMI-matched controls. We then examined whether FA oxidation from
endogenous lipid pools was compromised in the myotubes of these individuals with T2D. After an overnight loading of the myotube
lipid pool with oleate, no differences in endogenous FA oxidation rates between obese and T2D myotubes were observed. These
findings contrast those of Gaster (27), who demonstrated that complete oxidation of endogenous, but not exogenous, FAs was reduced in T2D myotubes compared with
those from obese control subjects. One major difference between the current study and Gaster is that we included subjects
who were marginally obese (although all had a BMI >30 kg/m2), resulting in a lower average BMI than that in Gaster. Another potential explanation is that these two studies compared
distinct subgroups of the obese population that may have been exposed to different environmental factors, which could uniquely
affect a varied genetic or epigenetic background. The current data suggest that a reduced myocellular fat oxidative capacity
in individuals with T2D may not be an intrinsic characteristic but, rather, a consequence of environment.

One bout of exercise has been shown to improve the capacity to store TAG in muscle and, thereby, prevent lipid-induced insulin
resistance (42). Moreover, by use of in vivo infusion of 13C-palmitate, Bergman et al. (44) showed that palmitate incorporation into skeletal muscle TAG was increased in endurance athletes compared with sedentary
control subjects. In the current study, we found that incorporation of palmitate into TAG but not into DAG was blunted in
skeletal muscle of individuals with T2D compared with BMI-matched controls and young, lean subjects. Collectively, these reports
indicate that increased channeling of FAs toward storage in the form of TAG in skeletal muscle is associated with an improved
metabolic profile. In contrast to the reduced oxidative capacity in the muscle tissue of individuals with T2D, which was not
retained in the myotubes, reduced incorporation of FAs into TAG was retained in the myotubes of individuals with T2D. By reducing
lipid intermediate accumulation, a high capacity to channel FAs toward intramyocellular neutral lipid (i.e., TAG) storage
may protect against lipid-induced insulin resistance. Thus, a reduced rate of TAG storage in T2D may be an important factor
in the development of lipid-induced insulin resistance in these individuals. In support of this hypothesis, an enhanced capacity
of the myotubes to incorporate endogenously labeled oleate into the total lipid pool, and specifically TAG, was associated
with reduced basal glucose concentration and insulin sensitivity.

The reason for the retained aberrant lipid incorporation capacity in T2D is so far unknown. We investigated whether skeletal
muscle FA uptake may underlie these findings. Skeletal muscle FA uptake is a controversial topic, with some investigators
finding no difference in uptake of FAs (7,8) and others showing increased FA uptake in skeletal muscle of obese and T2D individuals (9). In the current study, we could not detect differences in short-term FA uptake rates in cultured myotubes of individuals
with T2D compared with BMI-matched controls. Reduced TAG incorporation in the setting of unaltered FA uptake, oxidation, or
DAG incorporation might indicate that the FAs are incorporated into other lipid species not measured here that may (i.e.,
ceramides) (45) or may not yet be associated with insulin resistance and T2D. In addition, we could not detect differences in mRNA levels
of lipid metabolism genes or in enzymatic activity of DGAT between T2D and obese controls. However, we did observe significantly
positive associations of PGC1α mRNA with DGAT activity and the capacity to incorporate both palmitate and oleate into TAG
in the myotubes. These relationships support our previous findings that highlight the role of PGC1α in the regulation of intramuscular
lipid droplet programming in mice and humans (23). Finally, we focused on expression levels of the lipid droplet coat proteins PLIN5, PLIN3, and PLIN2 as well as the lipolytic
protein ATGL in both muscle tissue and myotubes because we have previously shown in animal studies that overexpressing PLIN2
or PLIN5 increases TAG storage capacity and results in the prevention of high-fat–induced insulin resistance (40,46). In the current study, we found that PLIN5 was elevated in the muscle tissue of the T2D individuals but not in their myotubes.
This finding is surprising given that the tissue lipid levels were not different between the two groups and that PLIN5 has
been associated with insulin sensitivity in humans (38). However, PLIN5 protein expression was not different between the two groups of myotubes in the basal state, which is in
line with the similar lipid levels observed in these myotubes. Moreover, PLIN5 protein levels increased after an overnight
oleate load in the BMI-matched myotubes but not in myotubes derived from T2D individuals. These data are consistent with the
increased neutral lipid and TAG storage observed in myotubes from the BMI-matched controls. In support of the notion that
an increased TAG storage capacity is protective against the development of insulin resistance in conditions of increased lipid
supply, we demonstrate that PLIN5 protein levels (postoleate load) in myotubes were positively related to insulin sensitivity.
In the context of similar lipid levels in the tissue and cells, it is not surprising that the expressions of PLIN2 and PLIN3
were not different between the two groups. However, when taken together, future studies should investigate the intrinsic pathways
in muscle lipid turnover (and its associated proteins) to identify the critical regulation sites.

In summary, the data show that skeletal muscle tissue lipid oxidation and FA incorporation into TAG are perturbed in obese
individuals with T2D compared with BMI-matched controls but that only the disturbances in TAG incorporation are conserved
in cultured myotubes from these individuals. The results are consistent with the view that lipid turnover has a significant
impact on insulin sensitivity and glucose homeostasis. Future studies using primary human muscle cell models with muscle-specific
modulations of the lipid turnover pathways may help to unravel the specific regulation sites of skeletal muscle and whole-body
energy metabolism in vivo. These findings could have profound implications for how we use precision medicine to treat, manage,
and prevent T2D in the future.

Article Information

Acknowledgments. The authors thank the study participants; Denis Dahlmans and Lucas Lindeboom of the Department of Human Biology, Maastricht
University, for assistance with the in vivo measurements; and Anne Gemmink of the Department of Human Movement Sciences, Maastricht
University, for assistance with the ex vivo measurements.

Funding. M.B. is financially supported by the NUTRIM—School for Nutrition, Toxicology and Metabolism and the Graduate School VLAG.
This work received support from Austrian Science Fund grant P25193 (to A.L.). This work also was partially funded by a Vici
grant (918.96.618) for innovative research from the Netherlands Organisation for Scientific Research (to P.S.).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. L.M.S. and M.B. researched data, contributed to the study concept and design and data analysis and interpretation, and wrote
the manuscript. B.B., T.v.d.W., and L.B. researched data and reviewed and edited the manuscript. G.S., E.M.-K., T.O.E., and
A.L. researched data. M.K.C.H. and P.S. contributed to the study concept and design and data analysis and interpretation and
reviewed and edited the manuscript. P.S. is the guarantor of this work and, as such, had full access to all the data in the
study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in poster form at the 73rd Scientific Sessions of the American Diabetes Association, Chicago,
IL, 21–25 June 2013.

. The endoplasmic reticulum enzyme DGAT2 is found in mitochondria-associated membranes and has a mitochondrial targeting signal
that promotes its association with mitochondria. J Biol Chem2009;284:5352–5361pmid:19049983